Alzheimer's disease (AD) is the most common neurodegenerative disease, which comprise a large group of pathologies caused by metabolic changes in brain cells, loss of synapses and other compartments of neurons, and finally neuronal death (for review see Neurodegenerative diseases: From Molecular Concepts to Therapeutic Targets. Editors: R. von Bernhardi, N.C. Inestrosa, Nova Publishers, 2008). Due to increased lifespan, neurodegenerative diseases in general and AD in particular have become very common in developed countries. In the US alone, there are currently more than 5.4 million (and 36 million worldwide) people living with AD, and estimated 70-80 million people, who are over 55 years old, are considered to be at risk of developing the disease. In 2011, the annual cost of healthcare services for AD patients in the US was estimated at $183 billion (Rocca, W. A. et al. Alzheimer's & Dementia. 2011, 7:80-93; http://www.alz.org/downloads/Facts_Figures_2011.pdf). Drug development and successful treatment of AD and other neurodegenerative diseases are significantly complicated by the absence of effective methods for their early diagnosis and monitoring. Development of effective diagnostic methods is further complicated by the strong brain potential to compensate for the dysfunction and loss of neurons over a long period of time. This results in late clinical manifestation of disease symptoms when treatment cannot be very successful due to serious morphologic changes in the brain including the massive loss of neurons. Thus, diagnostic methods based on detection of early events in the disease development are particularly desirable.
Alzheimer's disease is characterized by neuronal death in several disease-specific areas of the brain, such as hippocampus and cortex. However, the neuronal loss is a relatively late event in the disease progression that typically is preceded by synaptic dysfunction, synaptic loss, neurite retraction, and the appearance of other abnormalities such as axonal transport defects (See, e.g., Crews, Masliah, Human Mol Gen., 2010, 19:R12-R20; Bredesen, Molecular Neurodegeneration 2009, 4:27; Nimmrich and Ebert, Rev Neurosci. 2009, 20:1-12; Yoshiyama et al., Neuron. 2007, 53:337-351; Wishart et al., J Neuropathol Exp Neurol. 2006, 65:733-739; Gylys et al., Neurochem Int. 2004; 44:125-131; Conforti et al., Trends Neurosci. 2007, 30:159-166; Revuelta, et al. Am J Alzheimers Dis Other Demen 2008, 23: 97-102). Numerous studies are devoted to description of axon destruction with shedding of membrane-enclosed “axosomes”, axon, dendrite and spine pruning, and disassembly of synapses (Goda, Davis, Neuron 2003, 40:243-264; Eaton, Davis, Genes Development, 2003, 17:2075-2082; Koirala, Ko, Neuron, 2004, 44:578-580; Bishop et al., Neuron, 2004, 44:651-661; Low, Cheng, Phil. Trans. R. Soc. B 2006 361, 1531-1544).
Currently there are attempts to develop anti-AD therapeutics capable of restoring dendritic spine density and synapses (Adlard et al., PLoS ONE, 2011, 6:e17669).
The first symptomatic stage of Alzheimer's disease that is manifested by mild clinical symptoms is Mild Cognitive Impairment (MCI), which is usually defined as an intermediate state between normal aging and dementia (DeCarli, Lancet Neurol., 2003, 2:15-21; Stephan et al., Alzheimer's Res Therapy, 2009, 1:1-9; Apostolova et al., Human Brain Mapping, 2010, 31:786-797). On average, MCI patients convert to dementia at a rate of 10-15% annually (Petersen et al., Arch Neurol. 2001, 58:1985-1992; Apostolova et al., Human Brain Mapping, 2010, 31:786-797). However, currently the MCI outcome is not reliably predictable. First, up to 40% of MCI patients revert to normal status (Larrieu et al., Neurology, 2002, 59:1594-1599; Brooks, Loewenstein, Alzheimer's Res Therapy, 2010, 2:28-36), and autopsy studies demonstrate that a substantial percentage of MCI patients do not have evidence of AD pathology (Jicha et al., Arch Neurol, 2006, 63:674-681; Khan, Alkon, Neurobiol. Aging, 2010, 31:889-900). Second, about 20% of MCI patients who convert to dementia are diagnosed not with AD but other neurodegenerative diseases, such as vascular, Lewy body, Huntington, Parkinson, and other dementias (Jicha et al., Arch Neurol, 2006, 63:674-681; Stephan et al., Alzheimer's Res Therapy, 2009, 1:1-9). Third, disease progression varies for AD patients from slow to intermediate and rapid (Doody et al., Alzheimer's Res Therapy, 2010, 2:2-10). Even clinically MCI is not a homogeneous pathology and can be described as two conditions, with amnestic symptoms (aMCI) and without amnestic symptoms (Dlugaj et al., Dement Geriatr Cogn Disord., 2010, 30:362-373; Brooks, Loewenstein, Alzheimer's Res Therapy, 2010, 2:28-36). Some publications have demonstrated that aMCI converts to dementia much more often and is a better predictor of AD (Mariani et al., J Alzheimer's Dis., 2007, 12:23-35; Luck et al., Psychiatr Prax., 2008, 35:331-336; Koivunen et al., Neurology, 2011, 76:1085-1099). However, other authors have not found significant difference in the conversion rate for two MCI forms (Rountree et al., Dement Geriatr Cogn Disord., 2007, 24:476-482).
Currently, diagnosis of AD and other forms of dementia is based on analysis of the patient's cognitive function. As mentioned above, due to effective compensatory mechanisms in the brain, the decrease of cognitive function is usually registered when a disease is in its later stages and fewer treatments are available. Amyloid plaques between neurons, neurofibrillary tau-tangles, and an overall shrinkage of brain tissue are the hallmarks of AD, and there were many attempts to develop diagnostic tests based on these phenomena. New imaging techniques, including in vivo detection of β-amyloid deposition (e.g., positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), multiphoton imaging, magnetoencephalography (MEG), electroencephalography (EEG) etc.) (Mucke, Nature, 2009, 461:895-897; Mistur et al., J. Clin. Neurol., 2009, 5:153-166; Miller, Science, 2009, 326:386-389; Perrin et al., Nature, 2009, 461: 916-922) are becoming increasingly popular, but cannot be used for screening purposes.
The existing diagnostic molecular tests for AD and other forms of dementia can be divided into two groups. The first group is based on analysis of single nucleotide polymorphisms (SNP), which is helpful for predicting a higher risk of a disease but not for diagnostics (Bettens et al., Hum Mol Genet. 2010, 19(R1):R4-R11). The second group uses analysis of proteins involved in AD pathogenesis or brain-specific proteins, such as neural thread protein (NTP), in bodily fluids (Schipper, Alzheimer's & Dementia. 2007, 3:325-332). However, these tests are not sufficiently sensitive and specific. Recently published data have demonstrated high sensitivity of AD detection by measuring concentrations of three protein biomarkers (beta-amyloid protein 1-42, total tau protein, and phosphorylated tau181P protein) in the cerebrospinal fluid (CSF) (Meyer et al., Arch Neurol. 2010, 67:949-956; Fagan A. M. et al. Arch. Neurol, 2011, 68:1137-1144). The high invasiveness of the CSF collection procedure makes such tests impractical and challenging for everyday clinical use. Several groups have reported diagnostic assays for AD based on analysis of a large number of proteins or antibodies in human blood (Ray S. et al. 2007, Nat. Med. 13, 1359-1362; Reddy M. M. et al. 2011, Cell 144, 132-142; Nagele E. et al. 2011, PLoS One 6, e23112), However, other researchers were not able to confirm the results of these studies (Bjorkqvist M et al. 2012, PLoS One 7, e29868).
On the 19th of April, 2011 The National Institute on Aging/Alzheimer's Association provided new Diagnostic Guidelines for Alzheimer's Disease (Khachaturian Z S, 2011 Alzheimer's and Dementia. 7, 253-256). The new guidelines were published in four papers devoted to: (i) classification of the AD phases, namely the dementia phase, the symptomatic pre-dementia phase (MCI), and the asymptomatic, preclinical phase of AD (pre-MCI) (Jack et al., 2011, Alzheimer's and Dementia. 7, 257-262); (ii) Recommendations from NIA for the diagnosis of dementia due to AD (McKhann et al., 2011, Alzheimer's and Dementia. 7, 263-269); (iii) Recommendations from NIA for the diagnosis of MCI due to AD (Albert et al., 2011, Alzheimer's and Dementia. 7, 270-279); and (iv) Recommendations from NIA toward defining pre-MCI (Sperling et al., 2011, Alzheimer's and Dementia. 7, 280-292). The new guidelines stress the current lack of and a great need for reliable biomarkers which can be used for detection of pre-MCI and pre-symptomatic AD, as well as MCI and AD.
Thus, there is a huge need in a non-invasive or minimally invasive molecular test(s) capable to detect MCI or even earlier asymptomatic stages of AD (pre-MCI). Further, it would be even better if such a test could be used for prognosis of the disease outcome and disease and treatment monitoring.
Metabolic changes occurring in AD and other neurodegenerative diseases cause the destruction of spines, dendrites, axons, and synapse loss, and the latter likely induces neuronal death (Bredesen, Molecular Neurodegeneration 2009, 4:27; Crews, Masliah, Human Mol Gen., 2010, 19:R12-R20). Similar processes happen during embryonic brain development. Numerous neurons are trying to establish intercellular contacts, those neurons that do it successfully survive, and other neurons die (Butts et al., Cell Death Differ. 2008, 15:1178-1186; Enokido and Hatanaka, Gan To Kagaku Ryoho. 1994, 21:615-620; Gasic and Nicotera, Toxicol Lett. 2003, 139:221-227).
Axon destruction with shedding of membrane-enclosed “axosomes”, axon, dendrite and spine pruning, and disassembly of synapses lead to appearance of cell-free vesicles containing cytoplasmic components of neurons, axons, neurites, spines and synapses, including proteins, RNA and their degradation products. There are other processes leading to liberation of these compounds into the extracellular medium, in particular, blebbing (Charras et al., Biophys. J. 2008, 94:1836-1853; Fackler, Grosse, J. Cell Biol. 2008, 181:879-884), exocytosis (Skog et al. Nat Cell Biol., 2008, 10:1470-1476) and other forms of active secretion (Wang et al. Nucleic Acids Res., 2010, 38:7248-7259; Kosaka et al., J Biol Chem., 2010, 285:17442-17452; Pigati et al., PLoS ONE, 2010, e13515).
MicroRNAs (miRNAs) are a class of non-coding RNAs whose final product is an approximately 22 nt functional RNA molecule. They play important roles in the regulation of target genes by binding to complementary regions of messenger transcripts to repress their translation or regulate degradation (Griffiths-Jones Nucleic Acids Research, 2006, 34, Database issue: D140-D144). Frequently, one miRNA can target multiple mRNAs and one mRNA can be regulated by multiple miRNAs targeting different regions of the 3′ UTR. Once bound to an mRNA, miRNA can modulate gene expression and protein production by affecting, e.g., mRNA translation and stability (Baek et al., Nature 455(7209):64 (2008); Selbach et al., Nature 455(7209):58 (2008); Ambros, 2004, Nature, 431, 350-355; Bartel, 2004, Cell, 116, 281-297; Cullen, 2004, Virus Research., 102, 3-9; He et al., 2004, Nat. Rev. Genet., 5, 522-531; and Ying et al., 2004, Gene, 342, 25-28). There are other classes of less characterized small RNAs (reviewed in Kim, Mol. Cells, 2005, 19: 1-15).
Many of miRNAs are specific to or over-expressed in certain organs/tissues/cells (see, e.g., Hua et al., BMC Genomics, 2009, 10:214; Liang et al., BMC Genomics. 2007, 8:166; Landgraf et al., Cell. 2007, 129:1401-1414; Lee et al., RNA. 2008, 14:35-42).
Some miRNAs, including those that are cell-specific, are enriched in certain cellular compartments, particularly in axons, dendrites and synapses (see, e.g., Schratt et al., Nature. 439:283-289, 2006; Lugli et al., J Neurochem. 106:650-661, 2008; Bicker and Schratt, J Cell Mol Med., 12:1466-1476, 2008; Smalheiser and Lugli, Neuromolecular Med. 11:133-140, 2009; Rajasethupathy, Neuron. 63:714-716, 2009; Kye, RNA 13:1224-1234, 2007; Yu et al., Exp Cell Res. 314:2618-2633, 2008; Cougot, et al., J Neurosci. 28:13793-13804, 2008; Kawahara, Brain Nerve. 60:1437-1444, 2008; Schratt G. Rev Neurosci. 2009; 10:842-849; Pichardo-Casas et al. Brain Research. 1436:20-33, 2012).
Expression and concentrations of miRNAs are regulated by various physiological and pathological signals. Changes in expression of some miRNAs were found in neurons of Alzheimer's and other neurodegenerative disease patients (Hebert and De Strooper, Trends Neurosci. 32:199-206, 2009; Saba et al., PLoS One. 2008; 3:e3652; Kocerha et al., Neuromolecular Med. 2009; 11:162-172; Sethi and Lukiw, Neurosci Lett. 2009, 459:100-104; Zeng, Mol Pharmacol. 75:259-264, 2009; Cogswell et al., Journal of Alzheimer's Disease. 14: 27-41, 2008; Schaefer et al., J. Exp. Med. 204:1553-1558, 2007; Hebert, Proc Natl Acad Sci USA 2008; 105:6415-6420; Wang et al., J Neurosci. 2008, 28:1213-1223; Nelson et al., Brain Pathol. 2008; 18:130-138; Lukiw, Neuroreport. 2007; 18:297-300).
Due to their small size, miRNAs can cross the blood-brain, placental and kidney barriers. miRNA release can be activated by pathology, e.g. malignancy (Pigati et al., PLoS ONE, 2010, e13515). Analysis of cell/tissue-specific miRNAs in bodily fluids was proposed for detection of in vivo cell death (U.S. Patent Pub. No 20090081640; Laterza et al., Clin Chem. 2009, 55:1977-1983).
Cognitive function testing and brain imaging, which are currently used as main methods for diagnosis of neurodegenerative diseases such as AD, allow only detection of later stages of disease and are not sufficiently specific. There is still a great need in the art to develop methods for early diagnosis of MCI and AD prior to occurrence of major morphological changes and massive neuronal cell death.